Regulation of protein kinases Raf-1 and B-Raf by their cysteine -rich domains and by the small GTPases Ras and Rap1

We have investigated the differential regulation of Raf-1 and B-Raf, two protein serine/threonine kinases of the Raf family, by Rap1, a Ras-like GTPase. While Ras activates both Raf-1 and B-Raf, Rap1 inhibits the activation of Raf-1 but stimulates the activity of B-Raf. Since the cysteine-rich domain (CRD) is important for Raf-1 regulation, including inhibition by Rap1, we investigated potential functional difference(s) between the CRDs of Raf-1 and B-Raf. Mutational disruption of the CRD increases the basal activity of B-Raf determined after immunoprecipitation (IP) and in intact cells. However, Raf-1 containing a similar mutation exhibits reduced activity after IP but elevated activity in intact cells. Prominent structural differences between Raf proteins are irrelevant to the effects caused by the mutation in the CRD. Further analysis demonstrated that the disruption of the CRD causes both Raf proteins to lose the affinity of their N-terminal domains to bind with 14-3-3 isoforms. These results suggest that Raf proteins are similarly regulated by their CRDs and that their different response to Rap1 is unlikely due to a functional difference between their CRDs. Interestingly, mutation of the CRD blocks the Rap1 interaction with both Raf-1 and B-Raf, suggesting that Rap1 primarily interacts with the CRD in both Raf proteins. Consistent with these observations, mutation of the CRD abrogates the Rap1-mediated inhibition of Raf-1 and activation of B-Raf. Interestingly, substitution of tyrosine with aspartic acid at position 340 in Raf-1, which mimics the putative tyrosine phosphorylation state, allows Raf-1 to be activated, rather than inhibited, when co-expressed with Rap1, demonstrating that the Rap1-mediated effect is determined by the tyrosine phosphorylation state of Raf-1. These results, along with the fact that active Ras is usually located within the close vicinity of tyrosine kinases, strongly support a model in which Rap1 inhibits the Ras-mediated activation of Raf-1 by localizing Raf-1 to membrane sites where tyrosine kinases are not present. On the other hand, B-Raf can be activated by both GTPases because tyrosine phosphorylation and co-localization with activated tyrosine kinases are not required for its activation